Heparin is an heterogeneous, polydisperse, highly sulfated polysaccharide belonging to the family of glycosaminoglycans, made up of 1→4 linked disaccharide repeating units, consisting of an α-D-glucosamine (A) and a hexuronic acid, α-L-iduronic (I) or β-D-glucuronic (G) acid, with O-sulfate groups at different positions of the disaccharide unit, especially at position 2 of the iduronic (I2S) and position 3 and 6 of the glucosamine (A3S; A6S), and N-sulfate or N-acetyl groups at position 2 of the glucosamine residue(ANS; ANAc). The most frequently occurring repeating disaccharide sequence is (1→4)-α-L-iduronic acid-2-O-sulfate-(1→4)-α-D-glucosamine-N,6-disulfate (1→(I2S-ANS,6S), which represents the heparin highly sulfated segment, located closer to the non-reducing terminal of the heparin chain. Undersulfated sequences, accounted for by nonsulfated I and G, and ANAc are prevalently located toward the reducing end of the polymer. About one third of heparin chains contains a specific pentasaccharidic sequence, characterized by a central ANS,6S residue bearing an extra sulfate group at position 3 (ANS,3S,6S), constituting the active site for antithrombin III (AT). Many biochemical models as well as structural studies suggest that such pentasaccharide is located between the highly sulfated and the undersulfated domains. A minor sequence, involving neutral residues as galactose and xylose and corresponding to the reducing end of the polysaccharide chain, is the linkage region (LR) to the core protein of the proteoglycan.
The large number of possible structural variants of heparin sequences accounts for the wide range of biological activities that heparin promotes by binding to different plasma and tissue proteins, such as protease inhibitors of blood coagulation cascade, growth factors, chemokines, adhesive matrix proteins, etc. (Capila, & Linhardt, 2002). The identification of specific heparin structures responsible for binding to various protein ligands is of increasing interest. While some proteins, as AT, have affinity only for unique irregularities of heparin structure, others recognize the most regular regions of heparin, though this fact does not exclude selectivity of binding (Maccarana, Casu, & Lindahl, 1993).
Depending on their size and structural arrangement, heparin-derived oligosaccharides may elicit or inhibit specific biological effects. Typically, heparin sequences with a length ranging from tetra to decasaccharides are responsible for modulation of biological activity of proteins.
Different low and ultra low molecular weight heparin derivatives, ranging from 1900 to 4600 D, were recently demonstrated to cross the blood-brain barrier (BBB) in rats after oral or intravenous administration and to exhert a neuroprotective effect, potentially exploitable in the therapeutic treatment of neurodegenerative disorders. It is currently unclear what molecular weight fraction of these heterogeneous compounds crosses the BBB, and also what structural requirement is related to the biological action in the brain. The structural heterogeneity of heparin greatly influences the structure of the corresponding oligosaccharides. Moreover, each depolymerization reaction used for their preparation presents its own preferential selectivity, respect sequences and/or residues, and also usually modify at least the monosaccharide at the site of cleavage, generating further structural diversities.
U.S. Pat. No. 4,987,222 discloses a method for the depolymerization of heparin by the use of γ-rays. The examples disclose the preparation of heparin of average molecular weight by weight (Mw) around 5000 D and with a high S content. The patent shows a direct relationship between the amount of radiation and the reduction in Mw. However, the use of radiation according to U.S. Pat. No. 4,987,222 makes it possible only a limited reduction in Mw of heparin. Once a certain value of radiation is overcome, the colour becomes dark.
The degradation of heparin by γ-rays is strongly reduced when irradiating heparin in the presence of an organic compound as taught by WO 03/076474. The obtained depolymerised heparin is light in colour and does not require discolouration processes.
It has been surprisingly found that by combining at least two γ-ray irradiation processes with separation processes, it is possible to obtain heparin derived oligosaccharides having unique properties.